ILDR2 has a negligible role in hepatic steatosis.

We have previously reported that Ildr2 knockdown via adenovirally-delivered shRNA causes hepatic steatosis in mice. In the present study we investigated hepatic biochemical and anatomic phenotypes of Cre-mediated Ildr2 knock-out mice. Liver-specific Ildr2 knock-out mice were generated in C57BL/6J mice segregating for a floxed (exon 1) allele of Ildr2, using congenital and acute (10-13-week-old male mice) Cre expression. In addition, Ildr2 shRNA was administered to Ildr2 knock-out mice to test the effects of Ildr2 shRNA, per se, in the absence of Ildr2 expression. RNA sequencing was performed on livers of these knockdown and knockout mice. Congenital and acute liver-specific and hepatocyte-specific knockout mice did not develop hepatic steatosis. However, administration of Ildr2 shRNA to Ildr2 knock-out mice did cause hepatic steatosis, indicating that the Ildr2 shRNA had apparent "off-target" effects on gene(s) other than Ildr2. RNA sequencing and BLAST sequence alignment revealed Dgka as a candidate gene mediating these "off-target" effects. Ildr2 shRNA is 63% homologous to the Dgka gene, and Dgka expression decreased only in mice displaying hepatic steatosis. Dgka encodes diacylglycerol kinase (DGK) alpha, one of a family of DGKs which convert diacylglycerides to phosphatidic acid for second messenger signaling. Dgka knockdown mice would be expected to accumulate diacylglyceride, contributing to the observed hepatic steatosis. We conclude that ILDR2 plays a negligible role in hepatic steatosis. Rather, hepatic steatosis observed previously in Ildr2 knockdown mice was likely due to shRNA targeting of Dgka and/or other "off-target" genes. We propose that the gene candidates identified in this follow-up study may lead to identification of novel regulators of hepatic lipid metabolism.

ZNF70, a novel ILDR2-interacting protein, contributes to the regulation of HES1 gene expression.

A diabetes susceptibility gene, immunoglobulin-like domain containing receptor 2 (Ildr2), encodes a transmembrane protein localized to the endoplasmic reticulum membrane that is closely related to hepatic lipid metabolism. The livers of ob/ob mice in which Ildr2 is transiently overexpressed are relieved of hepatic steatosis. However, the molecular mechanisms through which ILDR2 affects these changes in hepatic lipid metabolism remain unknown. This study aimed to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underlying the role of ILDR2 in lipid homeostasis. We purified ILDR2-containing protein complexes using tandem affinity purification tagging and identified ZNF70, a member of the Kruppel C2H2-type zinc finger protein family, as a novel ILDR2-interacting protein. We demonstrated that ZNF70 interacts with ZFP64 and activates HES1 transcription by binding to the HES1 promoter. In addition, HES1 gene expression is increased in ILDR2-knockdown HepG2 cells, in which ZNF70 is translocated from the cytoplasm to the nucleus, suggesting that ZNF70 migration to the nucleus after dissociating from the ILDR2-ZNF70 complex activates HES1 transcription. These results support a novel link between ILDR2 and HES1 gene expression and suggest that ILDR2 is involved in a novel pathway in hepatic steatosis.

ILDR2: an endoplasmic reticulum resident molecule mediating hepatic lipid homeostasis.

Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. We used adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting "relief" of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, we investigated the possible participation of ILDR2 in ER stress responses. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. We conclude that manipulation of Ildr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time-course over which these studies were conducted, we cannot assign causal primacy to either the effects on hepatic lipid homeostasis or ER stress responses.

Absence epilepsy in apathetic, a spontaneous mutant mouse lacking the h channel subunit, HCN2.

Analysis of naturally occurring mutations that cause seizures in rodents has advanced understanding of the molecular mechanisms underlying epilepsy. Abnormalities of I(h) and h channel expression have been found in many animal models of absence epilepsy. We characterized a novel spontaneous mutant mouse, apathetic (ap/ap), and identified the ap mutation as a 4 base pair insertion within the coding region of Hcn2, the gene encoding the h channel subunit 2 (HCN2). We demonstrated that Hcn2(ap) mRNA is reduced by 90% compared to wild type, and the predicted truncated HCN2(ap) protein is absent from the brain tissue of mice carrying the ap allele. ap/ap mice exhibited ataxia, generalized spike-wave absence seizures, and rare generalized tonic-clonic seizures. ap/+ mice had a normal gait, occasional absence seizures and an increased severity of chemoconvulsant-induced seizures. These findings help elucidate basic mechanisms of absence epilepsy and suggest HCN2 may be a target for therapeutic intervention.

Positional cloning of "Lisch-Like", a candidate modifier of susceptibility to type 2 diabetes in mice.

In 404 Lep(ob/ob) F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lep(ob). The phenotypes of B6.DBA congenic mice include reduced beta-cell replication rates accompanied by reduced beta-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated "Lisch-like" (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646-amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes.

The genomic sequence of the accidental pathogen Legionella pneumophila.

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.